The effect of using pomegranate and strawberry juices with red beet puree on the physicochemical, microbial and sensory properties of yoghurt

There is currently a growing interest in creating dairy products fortified with natural fruits containing (phenolic and antioxidant compounds) and safe for human consumption. Therefore, this trend was applied in the current study, where a mixture of red beet puree with pomegranate and strawberry juices were used in the manufacture of yoghurt. The effect of supplementing yoghurt with the previous mixture on the physicochemical, microbiological properties, color, antioxidant activity, total phenolic content, reduced sugars, was studied. The use of fruit juices had slightly significant effect on the acidification rate of milk and the basic physical and chemical properties of the resultant yoghurt, but it increased slightly the red color. The total phenol content and the antioxidant activity were increased in yoghurt containing the mixture of fruit than in control one. Microbiological results revealed that the counts of lactic acid bacteria were increased in all treatments during storage and reached the maximum after 7 days of storage as slightly lower counts were recorded in the control treatment than in the other treatments, when fresh and during the storage period. All treatments were free from coliform and yeast and molds when fresh but it were noticed in all treatments at the 7 day. Organoleptic examination indicates that fruit treatments yoghurt gained the higher significant (p ≤ 0.05) scores as compared to control, when fresh and during storage period. Generally, results of this study show that the use of the mixture of the former fruits may be applied to produce a functional yoghurt rich in phenolic components, antioxidants activity and nutrients.


Introduction
Red beetroots have an excellent nutritional profile that includes plenty of essential vitamins, minerals and antioxidants. They also contain unique bioactive compounds called betalains, which may benefit a person's health [1], polyphenols, and powerful antioxidants. The health benefits of these chemicals have antidiabetic, anti-inflammatory, and anticancer activities [2]. The US Food and Drug Administration has approved beet extract (Betalain) as a color under code 73.40 [3], and the European Union has assigned it the designation E162 [4]. Betalains and polyphenols are unstable in the presence of light [5], high temperatures [6], an alkaline pH [7], enzymatic activity [8], and presence of oxygen and/or metals), alkaline pH, high temperatures, high temperatures, and high temperatures [9]. Their application in food has been limited because of their lack of stability [10]. A 100-mL serving of organic beetroot juice, contained 29 cal, no fat, 0.42 g protein, 7.5 g carbohydrates (5.42 g sugar, and 0.4 g fiber), iron, manganese, magnesium, zink, copper and selenium. It is good source of folate and potassium.
The pomegranate (Punica granatum L.), is a member of the Punicaceae family, is grown in a variety of microclimates throughout the world. Egypt, the United States, Turkey, Italy, India, Chile, Iran, and Spain are the countries that produce the most pomegranates [11]. Three million tons of pomegranates are produced worldwide each year. Pomegranate has important bioactive substances in many of its parts, including polyphenols, flavonoids, and anthocyanins, which have medicinal and functional properties, antioxidant properties, anticancer benefits, and anti-atherosclerotic effects. Because it contains both glucose and phenolic compounds, pomegranate juice is a nutritious beverage that is well-known for its sweet and tart flavor. The fresh juice contained 85.4% moisture, 10.6% total sugars, 1.4% pectin, 0.1 g/100 mL total acidity (as citric acid), 0.7 mg/100 mL ascorbic acid, 19.6 mg/100 mL free amino nitrogen and 0.05 g/100 mL ash [12].
Strawberries are a common and important fruit in the Mediterranean diet because of their high content of essential nutrients and beneficial phytochemicals, which seem to have relevant biological activity in human health. Among these phytochemicals, anthocyanin and ellagitannins are the major antioxidant compounds [13]. Fresh juice contained 91% moisture, 6.30% total sugar, 1.23% crude protein, 0.2% crude fat, 0.27% total ash, pH 3.3, 0.81% total soluble solids (T.S.S), and phenolic compounds 667.22 mg gallic acid equivalent in100g fruit juice (mg GAE/100 g of juice), total anthocyanine 47.65 (mg cyaniding glycoside/L).
Humans have been eating fermented foods for thousands of years, and they have been a vital part of their diet. Popular fermented milk product yoghurt is available in a wide range of flavors and shapes [14]. Yoghurt is a well-liked dairy product that contains alcohol as a result of a chemical reaction. It is used frequently for its nutritional value and other features that relate to vitamins, proteins, and other food components. Yoghurt's beneficial effects have been linked to the presence of (silver metal/ important nutrients), phosphorus, potassium, vitamins of high biological value, proteins, and vitally crucial fatty acids. While bifid tiny germ and Lactobacillus-enriched yoghurts are among the most popular varieties of functional foods, yoghurt is also a well-known probiotic carrier. Yoghurt consumption has been linked to numerous health benefits, including the prevention of weakening bones and weak bones, disease of the heart and blood vessels, and (a disease where blood sugar swings wildly), as well as (helping increase/showing positively) general gut health and controlling/adjusting the disease-fighting system [15]. Yoghurt consumption has risen quickly around the world for at least the past 20 years. Yoghurt recipes have (branched out/done different things) as a result of producers (ability to come up with interesting new things) and consumers (who use a product or services) demand healthier and tastier products, which has all led to the development of a range of products with various flavors, (states of health), and other characteristics. The use of fruit in yoghurt recipes is becoming popular [16]. The physicochemical (connected to vitamins, protein, etc.) and qualities of yogurt were significantly affected by the inclusion of fruits or fruit extracts [17]. These effects are peculiar to fruit and have to do with the fruit's food-like components and non-food-like components. For instance, berries are frequently added to yogurts as a source of phenolic chemicals [18]. It was well known that phenolic compounds interact with milk proteins to create protein-polyphenol complexes, which contribute to (relative to vitamins, protein, etc., in food) the qualities of phenolic compounds. This addition (to anything else) might result in the creation of new functional dairy products that could meet consumer desires (connected to products or services they use) [19]. It was observed that the total phenolic content (TPC) and total flavonoid content increased with pomegranate juice concentration [20]. Yoghurts flavored with pomegranate juice (PJ) had significantly higher antioxidant activity than the control, and it grew proportionately as pomegranate juice concentration rose. Pomegranate and strawberries juices can be used in place of sweets since they include the recommended daily amount of carbohydrates for an adult body as well as a significant amount of fiber and water [21], and help to cleanse and moisturize tissues.
One of the most common defects in yoghurts of the set kind is whey separation or syneresis. In the case of low-fat yoghurts or yoghurts made with the inclusion of fruit juices, this flaw could get worse [22].
This research aimed to evaluate the physicochemical, microbiological sensory characteristics, and antioxidant potential of cow milk yoghurt containing pomegranate juice, strawberry juice, and red beet puree.

Materials and methods
The experiment was conducted in the laboratory of the department of milk Animal Production Research Station in El-Karada, Animal Production Research Institute. Fresh cow's milk (acidity 0.16%, pH 6.6, moisture 87.5, fat 4.1, total protein 3.3%, Ash 0.75% and lactose 4.2%) was obtained from the herd of El-Karada Experimental Station. Animal Production Research Institute, Agricultural Research Center, Ministry of Agriculture. Whereas red beetroot, strawberry, and pomegranate were collected from a local market in Kafr Elshekh, Egypt. The yoghurt starter consisted of mixed strains of Streptococcus thermophilus and Lactobacillus delbrueckii subsp. bulgaricus was obtained from Chr. Hansen's Laboratories, Copenhagen, Denmark.
Preparation of red beetroot (RB) Puree: The Red beetroot are washed with clean water, and then the black spots are removed from them and cut into small cubes. Mix 1 kg of red beets with 200 mL of water and boil over the fire for 10 min, then cool and blend in the electric mixer. The resultant mixture is stored in plastic containers at a freezing temperature of − 18 °C until used. Preparation of pomegranate juice (PJ):

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Selection of fresh, undamaged pomegranate fruits (Punica granatum cv. Rabab), then fruits are manually peeled and crushed in electric blender (Ju2000 Vitae Moulinex, Barcelona, Spain). The resulting pomegranate juice (PJ) is filtered through cotton gauze to remove the remnants of the fruit and unwanted particles. And, then the resulting juice is stored at a freezing temperature (− 18 °C) until use [23]. It is added at a ratio of 0.5, 1, 1.5, and 2% of the weight of the milk after pasteurization and before inoculation with the starter. Preparation of strawberry juice: The strawberries are washed well and beaten in the electric mixer, then filtered with clean and sterile white gauze and stored at a freezing temperature (− 18 °C) the extract until used. It is added at a ratio of 0.5, 1, 1.5, and 2% of the weight of the milk after pasteurization and before inoculation with the starter. Yoghurt manufacturing: five treatments of yoghurt were made as follows: C: Control without any additives. T1: Yoghurt and 1% red beetroot puree + 0.5% strawberry juice + 0.5% pomegranate juice. T2: Yoghurt and 2% red beetroot puree + 1% strawberry juice + 1% pomegranate juice. T3: Yoghurt and 3% red beetroot puree + 1.5% strawberry juice + 1.5% pomegranate juice. T4: Yoghurt and 4% red beetroot puree + 2% strawberry juice + 2% pomegranate juice.
Cow's milk was heat-treated at 90 °C for 10 min, cooled to 45 °C, adding the puree and the different juices and 2% yoghurt starter. The resultant Yoghurts were incubated at 45 °C until complete coagulation, stored at 4 °C in the refrigerator until analyzes, when fresh and after, 3 and 7 days, for chemical, physical, microbiological and organoleptic properties.
Preparation of yoghurt water extract (YWE): yoghurt samples were diluted and homogenized at a 1:0.25 ratio with distilled water. To reduce the protein, the pH of the yoghurt solution was adjusted to pH 4.0 using 1.0 N HCl, incubated in a water bath at 45 °C for 10 min, centrifuged at 5000 rpm (4 °C), to remove precipitated milk proteins. After discarding the pellet, 0.5 N NaOH was added, to bring the supernatant's pH back down to 7.0, then second centrifugation at 5000 rpm for 10 min at 4 C was performed to further precipitate the proteins and salts [24]. The supernatant was used to analyze thereafter. Total solids, protein, fat, ash and vit. C contents and Titratable acidity were determined as described by [25]. The pH value was measured using pocket pH meter (IQ Scientific USA, Model IQ 125). The free radical DPPH · (2,2 diphenyl-1-picrylhydrazyl) technique [6] was used to determine the free radical-scavenging activity with few modifications. To be more precise, yoghurt water extract was diluted with distilled water to a total volume of 1.25 mL in a 5 mL Eppendorf tube (Eppendorf, Hamburg, Germany). 3.75 mL of DPPH solution was then added, vortexes, and let to stand at room temperature in complete darkness for 30 min. In a UV/Vis spectrophotometer (UV-1800, Shimadzu, Kyoto, Japan), the absorbance of samples and a blank (distilled water in place of the sample) were measured at 515 nm and quantified using Trolox as a reference.
With slight adjustments, the Folin-Ciocalteu technique [26] was used to quantify the total phenolic content. To be more precise, 1 mL of yoghurt water extract was diluted with 3 mL distilled water, then 250 µL of Folin-Ciocalteu reagent was added and vortexes in a 5 mL Eppendorf tube (Eppendorf). After one minute, 750 µL of 20% sodium carbonate (by weight) was added, vortexes and left at room temperature for 120 min while it was completely black. The absorbance was measured at 750 nm using a UV/V spectrophotometer (UV-1800, Shimadzu) and quantified using gallic acid as a standard.
The DNS (3,5-dinitrosalicylic acid) technique was used to identify reducing sugars [27]. In a glass test tube, distilled water was used to dilute the yoghurt water extract to a total volume of 500 µL, and added 500 µL of the DNS solution (1% w/v 3, 5-dinitrosalicylic acid, 30% w/v potassium sodium tartrate, 1.6% w/v Naoh). The test tube was then vortexes and heated in a water bath at 100 °C for five minutes. In the following step, 5 mL of distilled water was added when the tubes had cooled to room temperature. After vortexes the tubes, the absorbance was measured at 540 nm with a UV/V spectrophotometer (UV-1800, Shimadzu), and the amount was calculated using D-lactose as a standard.
The curd tension was measured by the method of [28]. The rate of curd syneresis was determined after 10, 30, 60, and 120 min at room temperature (25 ± °C) [29]. Viscosity (mPas) was a rotational viscometer (DV-III, Brookfield, MA, USA) with spindle no. 6 at the speed of 200 rpm [30]. Yoghurt samples were filled into 10 mL bottles and stored at (5 °C) during the storage period; the height of the supernatant was measured and divided its value by the total sample height in a bottle and expressed as the percent of serum separation [31].
The organoleptic properties were evaluated [36], with flavor scored from 60 points and body and texture 40 points. The organoleptic properties were assessed by 10 participants from the staff of the Animal Production Research Institute, Agricultural Research Center, Ministry of Agriculture.
All experiments were carried out in duplicate or triplicate samples were collected for each analysis. The threshold for significance was established at p < 0.05. The data were analyzed for statistical significance with ANOVA, and Tukey's honest significant difference (HSD) test was employed to determine significant differences among results; coefficients, ANOVA tables, and significance (p < 0.05) were computed using Statistica Version 5.0 (StatSoft Inc., Tulsa, OK, USA).

Results and discussion
The effect of using different levels of red beet puree, pomegranate and strawberry juices on total solids (TS), fat, protein, ash and vitamin C are illustrated in (Table 1). It can observed that significant increases (p ≤ 0.05) in TS, fat, protein, vitamin C and ash contents in yoghurt samples manifested with increased juice added. Treatments with red beet, pomegranate and strawberry juice (T3 and T4) showed a higher percent of total solids, fats, proteins and ash than the control. Vitamin C contents were higher in treatments contained fruit juices red beet, pomegranate and strawberry juice, and this behavior can be attributed to the fact that these fruits contain a high content of vitamin C. Vitamin C contents were 0.34, 1.7, 2.15, 2.96 and 3.15 mg/100 g for fresh control, T1, T2, T3 and T4 respectively.
As storage period progressed, there were noticeable and gradual increases in the contents of TS, fat, protein, ash and vitamin C. Table 2 shows that yoghurt's pH increased with increasing juice levels. The pH of yoghurt supplemented with juice ranged from 4.66 ± 0.01 to 4.75 ± 0.01 which is higher than the fresh control (pH 4.64 ± 0.03); this increase in pH could be attributed to the higher pH of the added ingredients to the yoghurts. In general, the pH values of all samples decreased during storage owing to increasing the acidity, and these differences were found to be significant (p < 0.05). These lower pH values can, also be explained by the further metabolic activities occurred by the starter cultures during storage resulting in decreasing the pH values, and increasing the aroma and acidic taste in the final product. The titratable acidity ranged from 0.85 ± 0.11 for T1 to 1.03 ± 0.01% for yoghurt treatment T2 (with 1% red beet puree, 0.5% strawbeery juice and 0.5% pomegranate juice). The lowest mean values of titratable acidity were found on the 1st day of storage, whearas the highest values were found on the 7 days. In the 1 day of storage, sample T2 had the lowest mean value (0.85%), whearas at the 7 days of storage T1 had the highest mean value (1.06%) of titratable acidity. This might be due to the higher rate of acid production in the experimental yoghurts during storage as a result of the fermentation of lactose by the action of the starter cultures. Similar results were reported by [37] for nonfat yoghurt.
The fermentation process was also monitored by measuring the remaining sugar in the milk. The addition of juices increased the initial sugar content of the milk. Control milk had a sugar content of 5.20% w/v and the addition of red beet puree, pomegranate and strawberry juices increased this value to 5.28, 5.40, 6.00, and 6.55 (% w/w lactose) for T1, T2, T3, and T4, respectively. A greater significant increase was observed in T4 (6.55% w/w lactose). These differences can be attributed to the high sugar content present in the additives. Storage affected the sugar content of all yoghurts, and despite previously reported differences in initial sugar content, final sugars were at (p < 0.05), ranging from 4.20 to 5.85 (% w/w lactose) among all treatments. Table 3 shows that the viscosity of yoghurt was affected by the proportions of juices added. Average viscosity values for all Yoghurt treatments increased with these addition being higher than those for the control yoghurt, probably because these additives had higher percent of total solids, fiber and sugars.
From the other side, these additives may alter the structure of the yoghurt's casein matrix in a way that contributed to the higher viscosity values. Increasing the fiber level in yoghurt preparation resulted in greater viscosity readings. The viscosity of yoghurt increased gradually with fruit additives until the 7 days of storage. Also, in Table 3, yoghurt fortified with fruit additives showed a decrease in serum segregation compared to the control. There was no serum separation in all fresh samples, but it was found slightly in stored samples.
It could be observed from Table 4 that the fortification of yoghurt with juices reduced its curd tension and increased its syneresis compared to the control. One of the most flaws  in fermented milk products, particularly set-type yoghurts, is the syneresis, this flaw may reduce the product's shelf life and affect consumer acceptance. Prior researches, linked between the addition of fruit juices to yoghurt's and increased syneresis and decreased curd tension. The introduction of red beet puree with pomegranate and strawberry juices increased the fiber content of the yoghurt, which would lock in water and thus increase the synergy. Watery composition of the juices themselves may cause more whey to be released into the yoghurt fortified with that juice. The higher synergy shown in the yoghurt is likely due to the higher active water content contributed by the added juices. The total phenolic content and antioxidant activity of yoghurts produced with the addition of the different juices are shown in Table 5. The total phenolic was significantly (p < 0.001) impacted by the usage of various juices. To be more precise, T4 showed the highest value whereas the control showed the lowest value. The addition of red beet, strawberry, and pomegranate juices had approximately similar values in treatments T1,T2 and T3, in spite it were significantly higher than the control. The fortification with juices increased the total phenolic compounds in fresh yoghurts from 55.30 (control) to 68.83 mg/100gm (T4). The presences of phenolic compounds in control yoghurt (41.6 mg/100 g) were expected because phenolic compounds are usually found in considerable amounts in ruminant milk. The majority of these compounds are derived from animal feed. During storage, a slight decrease was observed in all treatments. Indeed, the phenolic group is an excellent hydrogen donor that forms hydrogen bonds with the carboxyl group of the protein and, therefore, several studies have demonstrated that phenolic compounds may interact with proteins in several ways, both reversibly and irreversibly [38].
In vitro, the antioxidant activity of fresh yoghurt was measured using DPPH free radical scavenging activity and   Table 5). The same trend was observed as in the case of total phenols. Control yoghurt recorded the lowest percent of antioxidant activity, while the other treatments showed highest ones. A decrease in antioxidant activity was observed among all treatments, with storage for 7 days [23]. mentioned that yoghurt fortified with pomegranate juice have shown a tendency to increase antioxidant activity during storage. In general, interactions of phenolic compounds and proteins are known to influence on the antioxidant capacity, and bioavailability of phenolic compounds in foods [38].
Color is a very important characteristic of foods because it is the first sensory characteristic that consumers notice in foods and may influence their preferences. Color changes in yoghurt as a result of adding red beet puree and pomegranate, and strawberry juices were measured with L*, a*, b*, and chroma, Table 6. The L* values that explain the lightness of the product showed that adding juices significantly decreased (p < 0.05) the lightness of yoghurt. Average L* values of fresh yoghurts showed that control yoghurt was the lightest (95.50 ± 2.20), followed by T1 (92.35 ± 1.20) and T2, T3 (90.32 ± 1.10, 88.55 ± 1.22), and (85.30 ± 1.26) in a descending order. The same trend was observed in all treatments during storage. The addition of juices resulted in increasing a* value (redness) of yoghurts. More specifically, a* value of yoghurt was significantly increased (p < 0.05) from -3.50 ± 0.12 in control to 52.03 ± 0.52, 45.20 ± 0.42, 35.20 ± 0.22 and 32.70 ± 0.35 for T1, T2, T3, and T4, respectively, indicating an increase in the red color. Storage did not effect on a* value of the control. However, a slight decrease in the red color has been reported for the other treatments, at the end of storage time. The b* value (yellowness) of yoghurt was decreased by adding juices, especially in T4. Values of C* were also decreased for T1, T2, T3, and T4 after 7 days of storage. The degradation of BC leads to the formation of compounds with a yellow color and, this is reflected in the increase in the b* parameter [39].
Color saturation (Chroma) and hue (Hue angle) indicated differences (p < 0.05) between treatments and over time; however, treatments contained red beet did not show differences (p > 0.05). The color change (∆E) between treatments and days was also different (p < 0.05). Table 7 displays that the counts of lactic acid bacteria (LAB) in yoghurt were increased in all treatments during storage to reach the maximum counts after 7 days of storage. Control yoghurt recorded slightly lower counts than the other treatments, when fresh and during storage period. In the dairy industry, it is very important to facilitate the survival of yoghurt starter bacteria during the processing and storage of Yoghurts. Therefore, FAO/WHO has established a requirement of a minimum number of viable cells of yoghurt starter bacteria of 10 7 Cfu/G during consumption [40]. In the present study, these numbers were higher than 108 Cfu/G after 7 days of storage for all yoghurt Table 6 Color values L*, a*, b*, C*, h* and ∆E of yoghurt water extract, affected by affected by different levels of red beet pure and juices of strawberry, and pomegranate *See legend foot under Table 2 for more details L* = luminosity, a* = green (-) and red (+), b* = blue (−) and yellow (+), C* = chroma (color saturation), h* = Hue angle (hue), and ∆E = color difference  [41] evaluated how the addition of artichoke (Cynara scolymus), strawberry (Arbutus unedo L.), and cherry (Prunus avium L.) can affect the microbial properties of homemade yoghurts. They reported that poly-phenolic extracts had no significant effect on the microbial properties of yoghurt samples. Also, Jaster et al. [42] produced yoghurt with different levels of concentrated strawberry pulp (15% and 30%) and found that these additives cannot affect on the L. delbrueckii counts, and these counts maintained over 108 CFU/mL. Table 7 shows also that all treatments were free from coliform bacteria and yeast and molds when fresh. After that, yeast and molds were observed in all treatments and its counts were found slightly low in control yoghurt than the rest treatments. This may be due to the higher heat treatment applied and the hygienic condition of the production. These results are in line with those reported [36]. Table 8 indicates that treatments T1, T2, T3, and T4 (fruit juice yoghurt) gained the higher significant (p ≤ 0.05) scores for flavor and body& texture as compared to control when fresh and during storage. During storage, scores of flavor, body & texture were decreased with progress in the storage period. Treatment 3 was found the best and recorded the highest scores after 7 days of storage in the refrigerator.

Conclusion
The current study showed that the addition of red beet puree with pomegranate and strawberry juices in different proportions had slightly significant effect on the physical  and chemical properties of yoghurt except for the color (increased red color). On the other hand, the addition of these juices significantly increased the total phenolic content and antioxidant activity of the resulting yoghurt. These results are considered good for designing and developing new dairy products with increased functional properties. Further studies are needed to evaluate the potential changes of sensory properties, flavor compounds, and possible interactions may be done. In addition, this fruit yoghurt was useful in clarifying the potential health benefits of such products for consumers.
Funding Open access funding provided by The Science, Technology & Innovation Funding Authority (STDF) in cooperation with The Egyptian Knowledge Bank (EKB).

Conflict of interest
The authors declared that there is no conflict of interest.
Ethical approval This study does not present any ethical concerns.
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